Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport.

Mala V Rao,Takahiro Gotow,Nigel A Calcutt,Yasuo Uchiyama,Salvatore Mattina,Michael L Garcia,Yukio Miyazaki,Chris M Ward,Ralph A Nixon,Don W Cleveland,Aidong Yuan

doi:10.1083/jcb.200202037

Abstract

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine–serine–proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.

Highlights

Neurofilaments (NFs),* the most abundant structural component of large myelinated axons, are obligate heteropolymers (Ching and Liem, 1993; Lee et al, 1993) composed of neurofilament heavy (NF-H, 200 kD), neurofilament medium (NF-M, 160 kD), and neurofilament light (NF-L, 68 kD) subunits
neurofilament heavy subunit (NF-H) phosphorylation was markedly reduced in unmyelinated axonal segments, and this correlated with markedly reduced calibers, despite normal neurofilament content
To produce mice without the tail domain of NF-H (NFHtail⌬), a gene targeting vector was constructed from the mouse NF-H gene in which the COOH-terminal 612 amino acids of NF-H were replaced with a Myc epitope tag and a neomycin phosphotransferase gene (Fig. 1 A)

Summary

Introduction

Neurofilaments (NFs),* the most abundant structural component of large myelinated axons, are obligate heteropolymers (Ching and Liem, 1993; Lee et al, 1993) composed of neurofilament heavy (NF-H, 200 kD), neurofilament medium (NF-M, 160 kD), and neurofilament light (NF-L, 68 kD) subunits. NF-H tails do not control the rate of slow axonal transport | Rao et al 683 al., 1995; Tu et al, 1995; Marszalek et al, 1996; Wong et al, 1996; Xu et al, 1996) even in the presence of higher numbers of axonal neurofilaments (Monteiro et al, 1990; Xu et al, 1996). The COOH-terminal tail domains of NF-M and NF-H are phosphorylated at multiple lysine–serine–proline (KSP) repeat motifs. These phosphate groups appear on NF-M and NF-H only after they enter the axon (Sternberger and Sternberger, 1983; Lee et al, 1986; Glicksman et al, 1987; Oblinger et al, 1987; Nixon et al, 1987, 1990) and are metabolized relatively slowly compared to the NH2-terminal phosphates (Nixon and Lewis, 1986; Sihag and Nixon, 1991). NF-H phosphorylation was markedly reduced in unmyelinated axonal segments, and this correlated with markedly reduced calibers, despite normal neurofilament content

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Conclusion